ALBERT

Description: Abstract 3365 Background: A recent study suggested an increased rate of inhibitor development among Hispanic compared to White hemophilia A (HA) patients; however, a possible mechanism was not proposed or explored. In light of findings implicating a role for race-specific distributions of factor VIII (F8) and class-II human-leukocyte antigen (HLA-II) haplotypes on the disparate risk of inhibitor development in the Black HA population, we sought to determine the distributions of F8 and HLA-II haplotypes among Hispanic Americans and compare them to that of Black and White Americans. Methods: Using archived genomic DNA samples from the 194 unrelated founders of the San Antonio Family Heart Study (SAFHS), a collection of 1431 people from 41 large Mexican American families, we re-sequenced all exons of F8 to genotype the known nonsynonymous-single nucleotide polymorphisms (ns-SNPs) and identify any novel ns-SNPs. We then performed high-resolution HLA-II genotyping to identify each founders' pair of HLA-DRB1 alleles to compare them against those found in other ethnic groups. Results: Among the 291 potentially distinct Mexican American X-chromosomes evaluated, we identified the H2 F8 haplotype, defined by D1241E, in 25.0% of the subjects, which is in between that observed in the White (7.4%) and Black (37.4%) populations. We also found H3 F8, defined by D1241E and M2238V, in two subjects, who represent the first non-Black individuals reported to carry this haplotype. Furthermore, we discovered a previously unreported ns-SNP (H1919N), whose minor allele was found in only one male and defines a ninth wild-type F8 haplotype (H9). Regarding HLA-II alleles, the distribution among Mexican Americans in the present study was quite different from those found in Black and White individuals in the National Marrow Donor Program registry (see Figure). Three of the 4 most common HLA-II alleles in the Mexican Americans (DRB1*0802, DRB1*0407 and DRB1*1406) were seen in 〈 1% of both Blacks and Whites. Discussion: This is the first study to report the haplotypic prevalence of F8 and HLA-II alleles in Mexican Americans, the largest Hispanic ethnic group in the United States. By informing specific wild-type factor VIII (FVIII) peptides for use in HLA-II binding and T-cell stimulation assays, these results may help to identify high risk combinations of FVIII therapeutics and individual HLA-II repertoires that contribute to the higher rate of inhibitor development observed in Mexican versus Caucasian American HA patients. Disclosures: Viel: Histonis, Incorporated: Employment. Howard:Haplomics, Inc.: Equity Ownership.

Description: Intron-22-inversion patients express the entire Factor VIII (FVIII)-amino-acid sequence intracellularly as 2 non-secreted polypeptides and have a positive “intracellular (I)-FVIII-CRM” status. Mutations conferring a positive I-FVIII-CRM status are associated with low inhibitor risk and are pharmacogenetically relevant because inhibitor risk may be affected by the nature of the therapeutic FVIII-protein (tFVIII), the affinity of any tFVIII-derived foreign peptide (tFVIII-fp) for any HLA class-II isomer (HLA-II) comprising individual major histocompatibility complex (MHC) repertoires, and the stability of any tFVIII-fp/HLA-II complex. We hypothesize that mutations conferring a completely or substantially negative I-FVIII-CRM status are pharmacogenetically irrelevant because inhibitor risk is high with any tFVIII and individual MHC repertoire.

Description: Due to its position in the coagulation cascade, factor X (fX), a circulating zymogen is activated during hemostatic challenges to its serine protease fXa first via the extrinsic pathway’s tissue factor/factor VIIa complex and next by the intrinsic Xase complex of factors Ixa and VIIIa on phospholipids membranes. Among the congenital factor abnormalities manifesting as hemorrhagic predispositions, fX deficiency states, which are often autosomal recessive and associated with consanguinity, are among the rarest. Since concurrent studies on the unrelated Stuart and Prower kindreds in the southeastern United States and London (England), respectively, led to the discovery of fX, it is also known as the Stuart-Prower factor. Only 59 distinct fX gene mutations have been described worldwide since its discovery in the 1950s. We report a novel mutation within the encoding structural locus that we have designated FX-Augusta. The phenotype and genotype of the proband, a 14 year-old African-American boy, were studied and we are currently in the process of obtaining samples from the patient’s parents and siblings. The proband has experienced severe bleeding throughout his life with his first episode occurring three days after birth following circumcision. He experiences ~30 bleeds per year, mainly into joints and muscles, which occur spontaneously or following minor trauma. Although the patient appears to be mildly mentally retarded a formal workup has not occurred. On laboratory testing, a severely prolonged PT of 137.4 seconds and aPTT of 112.8 seconds were observed. While clotting-based fX activity (fX:C) levels were consistently 1kb of the flanking 3′-genomic DNA failed to identify consensus AAUAAA polyadenylation signals or variants of this highly conserved element. Second, the native fX 3′-end signals appear to be “strong” or efficiently utilized, as no alternative polyadenylation was found in a comprehensive review of the fX cDNAs in the NCBI database, which included 6 full-length cDNAs and 〉20 3′-ESTs containing polyA-stretches on the 3′-end of high-quality fX mRNA sequence. Finally, neither the causative 5-bp deletion nor the 8-bp insertion disrupts these native 3′-end signals. Nevertheless, the molecular studies underway, including fX:Ag measurements, 3′-end characterization of the mutant mRNA via 3′-RACE and western blotting, should help to distinguish between the possible molecular mechanisms.

Description: Abstract 1301 Poster Board I-323 Background Neutralizing factor VIII (FVIII) alloantibodies (“inhibitors”) develop in about 15% of all patients with hemophilia A following replacement therapy. The pathogenesis of this important treatment-related alloimmune complication is complex and involves both genetic and environmental factors, and likely their interactions. The type of hemophilia-causing FVIII gene (F8) mutation influences the risk of developing inhibitors with large deletions conferring the greatest risk and subtypes of single-base substitution mutations the lowest. Additional genetic factors that include possible transcription-modulating polymorphisms in the promoter regions of the structural loci encoding tumor necrosis factor a (TNFA), interleukin 10 (IL10), and cytotoxic T-lymphocyte associated protein-4 (CTLA-4) were recently found to be associated with alloimmunization risk in the Malmo International Brother Study (MIBS). We recently demonstrated that mismatched FVIII replacement therapy due to the presence of common F8 nonsynonymous-single-nucleotide polymorphisms (ns-SNPs), which result in single amino acid changes in FVIII but do not cause hemophilia, may represent a novel genetic determinant of risk and contribute to the greater frequency of inhibitor development in black compared to white patients with hemophilia A (Viel et al. N Engl J Med. 360:1618-27, 2009). Because the implicated immune response gene variants described above were not controlled for in our initial study, however, we are now evaluating their influence on inhibitor development in this sample of black Americans with hemophilia A. Methods We used PCR and genomic DNA from 78 black hemophilia A patients to genotype a SNP in the TNFA promoter (-308 G〉A) and a CA-dinucleotide repeat polymorphism in the IL10 promoter (IL10G) by direct sequencing and fragment-length analysis, respectively. The inhibitor status, baseline severity, age at enrollment, and biologic relationships of these study subjects were already known as was their hemophilic F8 mutations and the background haplotypes on which they reside. Analyses for genotype-specific associations with inhibitor status were performed with and without controlling for background F8 haplotype. Results Although we found the frequency of the 134bp IL10 risk allele (102bp in our modified assay) to be 20.3%, which is similar to that observed previously in the largely Euro-Caucasian MIBS cohort (26.8%), no obvious association between inhibitor risk and genotypes containing this allele was identified in the black hemophilia A patients studied, even after controlling for the background F8 ns-SNP haplotype. The frequency we identified for the -308A TNFA risk allele (8.8%) was similar to that observed previously in non-hemophilic African Americans (12%), but was far less frequent than observed in the MIBS (32.9%). Analogous to the IL10 results, we identified no association between inhibitor development and TNFA genotypes containing the risk allele (which was only G/A heterozygotes since no homozygous A/A individuals were identified) in this black patient cohort, even after controlling for their ns-SNP haplotypes. Conclusions We found no association between inhibitor development in black Americans with hemohilia A and promoter variants in two immune response genes recently implicated as risk genotypes in the MIBS. While we did not replicate the prior findings, the small sample size of our present study could explain these results if a true functional variant in one of these genes either has a small effect or is in poor linkage disequilibrium with the typed variant in this black patient population. Our findings therefore require confirmation in a larger study, which is currently under way. Disclosures Howard: Haplomics, Inc.: Equity Ownership, Patents & Royalties. Kempton:Biomeasure Inc: Consultancy; CSL Behring Foundation: Research Funding.

Description: Recombinant factor VIII (r-fVIII) is the most widely-used and effective therapy for hemophilia A (hA). Many patients unfortunately become refractory when the wildtype(wt) protein is seen as foreign and is targeted by functionally neutralizing anti-fVIII antibodies termed inhibitors. While inhibitors occur most often in severe hemophiliacs with complex fVIII mutations and a complete circulating absence of the fVIII protein, recent studies show patients with missense-mutations (mMt) have a higher incidence than previously thought and demonstrate r-fVIII can be immunologically targeted even when differing by only a single amino acid. Because mMt represent the most frequent overall etiology of hA (~39% of cases), common non-hemophilic protein variants of fVIII may represent an important novel modulator of inhibitor development in this setting. To determine the extent of such variation, we resequenced all coding regions of the fVIII loci (F8) in 137 healthy subjects from 7 ethnic groups, including 86 Caucasians and 16 African-Americans (AA). We identified 5 common nonsynonymous single nucleotide polymorphisms (nsSNPs). Surprisingly, whereas 4 were polymorphic in AA only 2 were variable in Caucasians, despite having examined 6x the number AA X-chromosomes. Since recent studies show AA patients have a 2-fold higher inhibitor incidence than Caucasians, thus establishing ethnicity as a risk factor in this complication, we were intrigued to find that minor alleles for 2 nsSNPs are restricted to AA and substitute amino acids in major B-cell inhibitor epitopes located in the A2 and C2 domains. To confirm these findings and accurately define the number and frequency of human haplotypes (H) (eg. distinct combinations in which the alleles of these 5 nsSNPs are linked in vivo) we resequenced F8 in a second study group that included 75 additional healthy AA. Here we show there are at least 7 distinct wt forms of the human fVIII protein by defining 7 haplotypes (H) from the 5 nsSNPs: H1, H2, …, H7. H1 exists in all ethnic groups, is the most common overall form of fVIII and represents 2 of the 3 r-fVIII concentrates used clinically. While H2 is the most common form in AA (44%) and possibly represents the other therapeutic r-fVIII molecule, we found that at least 20% will have AA-restricted fVIII proteins; H4 (4%), H5 (12%) and H7 (4%). Only 2 forms of fVIII were found in Caucasians, H1 (90%) and H2 (10%), in contrast, and neither were ethnically restricted. In summary, when combined with reports of at least 3 other nsSNPs, our findings establish as inaccurate the long held view that fVIII is basically a monomorphic protein in non-hemophiliacs. We hypothesize that greater immunologic barriers exist when r-fVIII is infused into patients with mMt in endogenous fVIII molecules containing one or more minor alleles of these nsSNPs. Moreover, due to the number and frequency of AA-restricted wt fVIII variants, we predict these nsSNPs contribute pharmacogenetically to the higher incidence of inhibitors in this ethnic group. As such, we have established a 6-site multi-center study to test this hypothesis using AA hA patients as the optimal group. So far we have enrolled 34 of 223 total AA hA patients at the participating centers and are obtaining blood samples from each for fVIII:C and fVIII:Ag measurements and F8 mutation detection. Because the allelic basis of hA has not been studied in AA, this mutation scan is essential to rule out the plausible alternative hypothesis that their higher inhibitor incidence is due to a distinct spectrum of molecular abnormalities. Ultimately, all patients with mMt will be tested to determine the presence and titer of anti-fVIII, using both Bethesda and ELISA assays, and the H of their mutant fVIII will be defined. The number of patients with inhibitors and AA-restricted forms (H4, H5 or H7) will then be compared to those with inhibitors but whose mMt are in haplotypes found in r-fVIII molecules (H1 or H2).

Description: Abstract 3366 Background/Aims: The hemophilia A (HA)-causing Factor (F)VIII gene (F8) mutation type is a well-established determinant of risk for the development of alloimmune inhibitors that neutralize replacement FVIII proteins in ∼20% of all HA patients. Studies have investigated variants of immune response genes to determine if they may account for the inter-individual variability in FVIII immunogenicity observed in patients with the same F8 abnormality, e.g., the intron-22 inversion. While some studies have found associations between inhibitor status and promoter polymorphisms in CTLA4, TNFA and/or IL10, others have not. If these promoter polymorphisms are indeed functional and truly influence inhibitor development, their alleles could modulate transcription initiation rates. The goal of this study was to investigate the possibility of cis-acting genotype-specific differences in mean steady-state mRNA levels encoded by CTLA4, TNFA and IL10. Methods: We examined the relationship of lymphocyte CTLA4, TNFA and IL10 mRNA levels with the genotypes of 265 SNPs located across their structural loci in 1189 Mexican American subjects in the San Antonio Family Heart Study. Expression profiles were generated using Illumina's HWG-6 BeadChips and genotypes came from the Illumina OmniExpress-12 BeadChip. Measured genotype association analyses that accounted for non-independence of family members and employed an additive model (in which testing to determine whether gene expression varies by genotype, with the model constrained so that each “dose” of the minor allele raises or lowers gene expression by an equal amount “beta”) were performed using the software package SOLAR. P-values were calculated using a 1 degree-of-freedom chi-square test comparing the likelihood of a model where the change in expression levels by genotype is estimated to the likelihood of a model where beta is constrained to zero. Results: None of the 265 genotyped SNPs within or near these three genes (i.e., 49 SNPs in IL10, 35 SNPs in CTLA4 & 181 SNPs in TNFA) function as cis-acting regulatory variants, as no significant genotype-specific associations with these genes' transcript levels were identified. Conclusions: We observed no evidence for cis-regulation of CTLA4, TNFA or IL10 in Mexican Americans, the largest and most rapidly growing minority population in the United States, despite having genotyped directly the previously implicated promoter polymorphisms in the current analysis (e.g., see Figure). Although Hispanic American HA patients were recently found to have a significantly higher risk for inhibitor development than White HA patients, it is possible that cis-acting functional variants in this minority population are rare and not well-represented by the common GWAS SNPs used for these analyses. Since linkage disequilibrium patterns between markers are population-specific, we are also currently genotyping these SNPs in a large cohort of African and Caucasian American HA patients. Disclosures: No relevant conflicts of interest to declare.

Description: The development of neutralizing antibodies-termed "inhibitors"-to infused therapeutic (t) factor VIII proteins (tFVIIIs) is the most serious obstacle to effective treatment of bleeding in Hemophilia A (HA) patients. As clinically significant FVIII immune responses are only initiated if dendritic cell (DC) cII-HLAs can present foreign tFVIII-derived peptides to naïve FVIII-specific T cells, we posit the "Gate Keeper" hypothesis in which the limiting determinant of inhibitor formation are patients' cII-HLA repertoires with the majority being individually distinct and each contributing slightly to the vast population level diversity of cII-HLAs. While cII-HLAs are critical at the cellular level for initiating immune responses, conflicting results from population studies have led some to describe their encoding HLA-II structural genes as weak determinants of inhibitor causation. Our main objective here is to test a hypothesis that gets at the heart of this disconnect between molecular-based expectations and population-level data by analyzing cII-HLA peptidomic data from DC-protein processing and presentation assays (PPPAs). The chief variable of DC-PPPA data is the peptide count, which we assume to be directly proportional to immunogenic potential (IP). Our working model is that inhibitor formation requires at minimum, in its initial stages, a complex between cII-HLAs and specific tFVIII-derived peptides. A testable null hypothesis under this thinking posits that a given cII-HLA allotype will have the same IP when exposed to several tFVIIIs. To test this hypothesis, we first performed model selection to determine the best set of predictor allotypes. To analyze the data, we employed a log-linear model where the peptide count is the dependent variable and allotype is a categorical independent variable consisting of 29 levels for 29 allotypes (8 DP, 10 DQ, and 11 DR allotypes). We used elastic net regression (ENR) to select the best set of allotype levels thus giving the best overall model consisting now of only four DR allotypes (Table 1). We then performed interaction analysis under the best-selected allotypes model in which we introduced as additional predictor variables, a tFVIII categorical variable consisting of five levels for five different tFVIIIs, namely full length (FL)-recombinant (r) FVIII (FL-rFVIII) ± von Willebrand Factor (VWF), B domain truncated (BDT)-rFVIII ± VWF, and plasma derived (pd) FVIII (pdFVIII) + VWF, and 12 interaction terms for the (4 - 1) × (5 - 1) possible interactions between the cII-HLA allotype and tFVIII variables. We found significant cII-HLA allotype × tFVIII interactions (Table 2). To get at the specific null hypothesis of interest, we examined within-allotype risk ratios (RRs) and their appropriately adjusted confidence intervals (CIs).1-4 It can be shown that an 84% CI is sufficient to achieve a significance level of α = 0.05 for the CI difference.2-4 Although there are 12 total interaction terms, per allotype there are only three possible CI comparisons on using the interaction term with the highest RR as a fixed reference. On constructing the adjusted CIs and correcting for multiple hypothesis testing,2 we found that two comparisons in Table 2 corresponded to significantly different RRs. We determined statistical power to detect a CI difference.1,3 As seen in Table 2, our study was extremely underpowered, which may explain why only two significant differences were found. Thus, at least for the two comparisons showing significant difference, we have refuted the null hypothesis of no difference across tFVIIIs for a given allotype, and have affirmed our working model that specific combinations of cII-HLAs and tFVIII-derived peptides are the triggering factor in inhibitor development.Schenker N, Gentleman J. On judging the significance of differences by examining the overlap between confidence intervals. Am Statistician. 2001; 55(3): 182-6.Julious S. Using confidence intervals around individual means to assess statistical significance between two means. Pharmaceut Statist. 2004; 3: 217-22.Maghsoodloo S, Huang C-Y. Comparing the overlapping of two independent confidence intervals with a single confidence interval for two normal population parameters. J Statist Plan & Infer. 2010; 140: 3295-305.Knol M, Pestman W, Grobbee D. The (mis)use of overlap of confidence intervals to assess effect modification. Eur J Epidemiol. 2011; 26(4): 253-4. Disclosures Hofmann: CSL Behring: Employment. Dinh:Haplomics Biotechnology Corporation: Employment, Equity Ownership. Escobar:Pfizer: Research Funding; Bayer, CSL Behring, Genentech, Hemabiologics, Kedrion, Novo Nordisk, Octapharma, Pfizer and Shire: Consultancy. Maraskovsky:CSL Behring: Employment. Howard:CSL Behring: Research Funding; Haplomics Biotechnology Corporation: Equity Ownership, Other: Chief Scientific Officer, Patents & Royalties: Patent applications and provisional patent applications .

Description: Abstract 39 Neutralizing anti-factor VIII (FVIII) antibodies, or “inhibitors”, interfere with FVIII pro-coagulant activity, and persistent inhibitors can result in significant morbidity and mortality in hemophilia A (HA) patients and in individuals who develop autoantibodies to their endogenous FVIII. Inhibitor production follows stimulation of helper T cells by linear amino acid sequences in FVIII corresponding to HLA-restricted T-cell epitopes. An immunodominant HLA-DRB1*01:01-restricted T-cell epitope within a peptide corresponding to FVIII residues 2194–2213 was identified previously using blood samples from two mild HA subjects with hemophilic mutation A2201P. This same immunodominant epitope was found recently in inhibitor subjects with (a) a large F8 gene deletion and b) an F8 nonsense mutation in exon 12. The present study aims to identify amino acid substitutions in FVIII that will neutralize this T-cell epitope while preserving the pro-coagulant activity of the modified FVIII protein. MHC class II - peptide binding assays were carried out using truncated FVIII peptides to determine the shortest sequence with full binding affinity for a recombinant HLA-DR0101 protein, and subsequently using peptides having systematic arginine substitutions at each position to identify the specific residues that confer this binding affinity. The results indicated that FVIII2194–2205 is the minimal binding epitope and that residues F2196, M2199, A2201 and S2204 interact with the peptide-binding groove of HLA-DR0101. Next, four T-cell clones that all proliferate in response to this epitope but have different T-cell receptors were stimulated with 12 peptides having systematic alanine substitutions at each position of FVIII2194–2205. The F2196A substitution abrogated proliferation of all four clones. The M2199A-substituted peptide stimulated three of the clones more weakly than the wild-type peptide. Peptide binding and T-cell assays were next carried out with FVIII2194–2205 peptides in which the 19 common non-phenylalanine amino acids were substituted at position 2196. These results identified 12 different amino acid substitutions that decreased both MHC binding and T-cell proliferation more than 10-fold. The binding of FVIII2194–2205 and FVIII2194–2205, F2196A to 10 common HLA-DRB1 proteins was measured to determine the potential promiscuity of this epitope. Moderate or low affinity binding of FVIII2194–2205 (IC50 〈 50 mM) to DR0401, DR0404, DR0901, DR1001, and DR1501 was observed. FVIII2194–2205, 2196A did not bind to any of the HLA-DRB1 proteins, suggesting that this substitution would not introduce a neo-epitope recognized by these other common MHC class II receptors. A recombinant FVIII-C2 domain protein with substitution F2196A was generated in E. coli and purified to homogeneity following a procedure that removes endotoxin. This FVIII-C2 mutein failed to stimulate the same four T-cell clones, all of which showed a strong, dose-dependent response to wild-type FVIII-C2. Recombinant B-domain-deleted FVIII (BDD-FVIII) proteins with substitutions F2196A, F2196L, F2196K, M2199A, M2199W and M2199R were expressed in BHK-M cell lines. Multiple cell lines were generated to express wild-type BDD-FVIII and each of these mutant proteins. Expression levels of the muteins were similar to that of wild-type BDD-FVIII except for the M2199W and F2196A variants, which had expression levels ∼30 and 10% that of wild-type BDD-FVIII, respectively. Specific activities of the muteins, measured using chromogenic and clotting assays, were similar to that of wild-type BDD-FVIII. Binding of these muteins to plasma-derived von Willebrand factor was evaluated by ELISAs, as a surrogate assay to indicate possible effects of specific mutations on FVIII half-life in the circulation. Their affinities for VWF ranged from ∼40–100% that of wild-type BDD-FVIII. Our results suggest that FVIII muteins with amino acid substitutions that abolish binding to DR0101 and retain reasonable FVIII functionality could be developed as less immunogenic therapeutic proteins, in order to avoid HLA-DRB1*01:01-restricted immune responses in HA patients with this common allele. The immunogenicity of this T-cell epitope and of the sequence-modified peptides and proteins in HA subjects with other HLA-DRB1 alleles is currently under investigation. Disclosures: Pratt: Puget Sound Blood Center: Employment, patent describing design of novel factor VIII proteins Other.

Description: Introduction: Factor (F)VIII immunogenicity is the main obstacle to both successfully treating hemophilia and receiving FDA approval for new therapeutics. Since immune responses to allogeneically distinct proteins require T-cell activation and proliferation, at least one foreign peptide must be liberated from FVIII that can bind with high affinity to one or more of a patient's HLA-class-II (HLA-II) isomers. Due to the complex pathogenesis of inhibitor development, which involves numerous genetic variations and both treatment and product related variables, our ability to predict which patients will become alloimmunized to FVIII remains suboptimal. The role of FVIII glycosylation in inhibitor development appears to be an important, yet not well characterized, modifying influence; defining the mechanism has been complicated by the fact that some FVIII products are highly glycosylated with thousands of potential glycoforms. Here we describe studies of the spectrum of FVIII peptides eluted from HLA-II complexes and the impact of glycosylation on the proteolysis and/or binding of these peptides to a patient's individual HLA-II isomers, i.e. to further incorporate personalized medicine in hemophilia care. Methods: Immature monocyte-derived dendritic cells (DCs) from 12 unrelated donors were generated in vitro and matured in the presence of an equimolar pool of a full length (FL) and 4 B-domain deleted (BDD) rFVIII proteins free of VWF and other proteins. Following harvest and lysis, HLA-DP, -DQ, and -DR molecules were recovered using a specific immunoaffinity step. Peptides were then eluted from these complexes and sequenced by high resolution mass spectrometry (LC/MS/MS). The bound peptides were mapped to the FVIII reference sequence. The estimated binding affinity of FVIII peptides to the HLA-II alleles found in these donors were obtained from NetMHCIIpan and compared to the peptides observed to be bound. Results: The 12 DC donors express 29 distinct HLA-II isomers (6 DP, 11 DQ, 12 DR) from an overall HLA-II gene repertoire having the following alleles: DP: 01:03/02:01, 01:03/03:01, 01:03/04:01, 01:03/04:02, 02:01/01:01, 02:02/05:01 DQ: 01:01/05:03, 01:02/06:02, 01:02/06:09, 01:03/05:01, 01:03/06:03, 01:03/06:04, 02:01/02:01, 02:01/02:02, 03:01/03:02, 03:02/03:03, 05:01/03:01 DR: 01:01, 03:01, 04:01, 04:04, 07:01, 09:01, 11:01, 11:04, 13:01, 13:02, 14:01, 15:01 These 29 HLA-II isomers were found to have 77 bound peptides that covered 1,202 of the 2,332 total amino acid residues in the NCBI reference FL-FVIII protein, whose domain structure and consensus N-linked glycosylation (NLG) sites are shown in panel A of the Figure. The HLA-II bound peptides were mapped to the FVIII regions from which they derive (panel B). One of the 20 NLG sites known to be glycosylated and 3 of the 4 known non-glycosylated NLG sites were found to be in a bound peptide. A NetMHCIIpan analysis predicted that no FVIII nonamer containing a consensus NLG site binds strongly to any of the 12 DR isomers; but, it also clearly showed that more peptides bind with weak or strong affinity as peptide length increases (data not shown). Conclusion: Despite finding 3 of FVIII's 4 non-glycosylated consensus NLG sites in the tightly bound HLA-II/peptide complexes, only 1 of the 20 glycosylated NLG sites was found among these bound peptides. The near complete absence of N-linked glycans in the presented HLA-II bound peptides is compelling, but incomplete, evidence that glycosylation influences the immunogenicity of FVIII. Potential mechanisms may include effects on internalization, proteolysis and/or HLA-II binding. Further direct studies are required to determine if these post-translational modifications affect proteolysis and/or HLA-II binding; core peptides that can be proteolyzed and/or bound in the absence of glycosylation or with alternative glycan conformations may provide more evidence that glycosylation can modulate immunogenicity. We hypothesize that HLA-II bound and unbound FVIII peptides constitute a novel immune-response-related biomarker that will improve the accuracy of inhibitor risk prediction by allowing pertinent patient-specific pharmacogenomic inputs to be analyzed in a more biologically relevant manner. References: Karosiene et al. NetMHCIIpan-3.0, a pan-specific MHC-II prediction method including all 3 human MHC-II isotypes: HLA-DR, -DP and -DQ. Immunogenetics, 2013. Disclosures Hofmann: CSL Behring: Employment. Zollner:CSL Behring: Employment. Powell:CSL Behring: Employment. Maraskovsky:CSL Behring: Employment. Howard:Baxter: Consultancy, Honoraria, Research Funding; Bayer: Consultancy, Honoraria, Research Funding; Haplomics, Inc.: Patents & Royalties; CSL Behring: Consultancy, Honoraria, Research Funding.